An analysis of the tRNA population of mouse mammary tissue showed very similar patterns of fractionation of isoacceptors during development and in response to prolactin in mouse mammary explants. Two minor seryl-tRNAs from mammalian cells suppress the nonsense codon UGA in protein synthesis. They are the first authentic nonsense suppressor tRNAs found in mammals. Furthermore, they form phosphoseryl-tRNA in the presence of an extract from lactating mammary tissue. The phosphoseryl-tRNAs recognize UGA in a ribosome-binding assay which suggest that these isoacceptors donate phosphoserine directly to protein.